Specific binding of 11-ketotestosterone in an androgen target organ, the kidney of the male three-spined stickleback,Gasterosteus aculeatus

Abstract

The kidney of male three-spined stickleback,Gasterosteus aculeatus, hypertrophies during the breeding season and produces a "glue" which is used in the building of the nest. This hypertrophy is androgen dependent, with 11-ketotestosterone (11KT) being more effective than other tested steroids in stimulating this secondary sexual character. In the present study kidneys were excised from stickleback males that had been castrated two days earlier. The purpose of this gonadectomy was to reduce the endogenous levels of androgens without allowing time for the kidney to regress. Tissue fragments were incubated with tritiated 11KT with and without unlabelled steroids at increasing concentrations. Displaceable specific 11KT binding was found in kidney tissue fragments whereas only non-specific binding was observed when liver and muscle were investigated in a similar way. Unlabelled 11KT displaced specifically bound, tritiated 11KT with an ED50-value (50% of displaceable binding) of 28 nM. Similar ED50 values were found for 17\-hydroxy-5α-androstane-3,11-dione (29 nM) and 5α-dihydrotestosterone (20 nM), whereas higher ED50 concentrations were estimated for testosterone (T; 203 nM) and progesterone (69 nM). No displacement of tritiated 11KT was found for the other investigated substances tested; estradiol, 17α,20β-dihydroxy-4-pregnen-3-one, flutamide or cyproterone acetate. No specific binding to kidney tissue fragments could be detected when labelled T was used instead of labelled 11KT. Specific binding of 11KT or T was not found either in the kidney cytosol or nuclear extracts. However, using the kidney membrane fraction a displacement of tritiated 11KT with unlabelled 11KT (10(-6)M) was observed. In conclusion there is a specific binding of 11KT in the stickleback kidney. The absence of binding in liver and muscle, the ED50 value observed and the displacement with some, but not all steroids are consistent with a receptor function. The presence of binding in membrane fractions, but not in cytosol or nuclear extracts suggests that the binding is not related to classic steroid receptors.

Use of the Three-Spined Stickleback ( Gasterosteus aculeatus ) As a Sensitive in Vivo Test for Detection of Environmental Antiandrogens

“…In the stickleback kidney, previous studies have shown that no specific binding of 11-ketotestosterone (11-KT) or testosterone was detected in either cytosolic or nuclear fractions, although displacement of tritiated 11-KT with unlabeled 11-KT was observed in the kidney membrane fraction (Jakobsson et al 1996). More recently, molecular cloning of a nuclear AR in the stickleback kidney has revealed that it is the classic mammalian type, AR2 or AR beta (GenBank accession no.…”

Detection of environmental androgens: A novel method based on enzyme‐linked immunosorbent assay of spiggin, the stickleback (Gasterosteus aculeatus) glue protein

“…However, preliminary results show that spiggin induction by 17α‐MT and DHT can be inhibited by the antiandrogenic drug flutamide [26], suggesting that these steroids are acting directly on a receptor (or receptors). In their studies on androgen binding by stickleback kidneys, Jakobsson et al [27] found no binding of 11‐KT or testosterone to the cytosolic and nuclear fractions, which is where the receptors in mammals are found. However, they did observe binding of 11‐KT to the membrane fraction, suggesting this as the location of the AR, but this suggestion contrasts with findings reported for other fish [28–30].…”

Differential binding of endogenous steroids and chemicals to androgen receptors in rainbow trout and goldfish

“…Pasmanik and Callard [8] also failed to detect specific binding with the ligand [ 3 H]11‐keto‐testosterone in goldfish brain ARs. Specific binding of [ 3 H]11‐ketotestosterone was observed in stickleback kidney membrane fractions, but was not found in cytosolic or nuclear fractions, suggesting the binding was not related to classic steroid receptors [26].…”